Bottom Line:
We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes.Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro.We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

ABSTRACTThe Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

Figure 9: Deubiquitination of the ubiquitinated AF-6 by Fam-CAT. AF-6 and HA-tagged ubiquitin (HA-Ub) were expressed with or without Fam-CAT (the deubiquitinating catalytic domain of Fam) in COS7 cells. The immunoprecipitates were then collected with anti-AF-6 antibody (#3), and subjected to an immunoblot analysis with anti-HA antibody or anti-AF-6 antibody (#4). The arrowhead denotes the position of HA-Ub–conjugated AF-6, and the arrow denotes the position of the immunoprecipitated AF-6. The results shown are representative of three independent experiments.

Mentions:
For the determination of whether Fam can release ubiquitin from the ubiquitinated AF-6 in vivo, AF-6 and HA-Ub were expressed with or without Fam-CAT in COS7 cells. We used Fam-CAT, which includes the deubiquitinating catalytic domain of Fam (1210–2410 aa), because we could not detect the expression of full-length Fam, probably due to its low expression level. As described above, AF-6 was immunoprecipitated from the cell lysates with anti-AF-6 antibody, and the immunoprecipitates were subjected to an immunoblot analysis with anti-HA antibody. In the absence of Fam-CAT, HA-Ub–conjugated AF-6 was observed (Fig. 9) as shown in Fig. 8. When AF-6 was coexpressed with Fam-CAT, the amount of HA-Ub–conjugated AF-6 was reduced while the amount of immunoprecipitated AF-6 was not affected (Fig. 9). These results indicate that Fam inhibited the ubiquitination of AF-6, and suggest that AF-6 is one of the substrates of Fam.

Figure 9: Deubiquitination of the ubiquitinated AF-6 by Fam-CAT. AF-6 and HA-tagged ubiquitin (HA-Ub) were expressed with or without Fam-CAT (the deubiquitinating catalytic domain of Fam) in COS7 cells. The immunoprecipitates were then collected with anti-AF-6 antibody (#3), and subjected to an immunoblot analysis with anti-HA antibody or anti-AF-6 antibody (#4). The arrowhead denotes the position of HA-Ub–conjugated AF-6, and the arrow denotes the position of the immunoprecipitated AF-6. The results shown are representative of three independent experiments.

Mentions:
For the determination of whether Fam can release ubiquitin from the ubiquitinated AF-6 in vivo, AF-6 and HA-Ub were expressed with or without Fam-CAT in COS7 cells. We used Fam-CAT, which includes the deubiquitinating catalytic domain of Fam (1210–2410 aa), because we could not detect the expression of full-length Fam, probably due to its low expression level. As described above, AF-6 was immunoprecipitated from the cell lysates with anti-AF-6 antibody, and the immunoprecipitates were subjected to an immunoblot analysis with anti-HA antibody. In the absence of Fam-CAT, HA-Ub–conjugated AF-6 was observed (Fig. 9) as shown in Fig. 8. When AF-6 was coexpressed with Fam-CAT, the amount of HA-Ub–conjugated AF-6 was reduced while the amount of immunoprecipitated AF-6 was not affected (Fig. 9). These results indicate that Fam inhibited the ubiquitination of AF-6, and suggest that AF-6 is one of the substrates of Fam.

Bottom Line:
We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes.Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro.We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

ABSTRACTThe Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.